Transannular Disulfide Formation in Gliotoxin Biosynthesis and Its Role in Self-Resistance of the Human Pathogen Aspergillus fumigatus

Abstract

Gliotoxin (1), the infamous representative of the group of epipolythiodioxopiperazines (ETPs), is a virulence factor of the human pathogenic fungus Aspergillus fumigatus. The unique redox-sensitive transannular disulfide bridge is critical for deleterious effects caused by redox cycling and protein conjugation in the host. Through a combination of genetic, biochemical, and chemical analyses, we found that 1 results from GliT-mediated oxidation of the corresponding dithiol. In vitro studies using purified GliT demonstrate that the FAD-dependent, homodimeric enzyme utilizes molecular oxygen as terminal electron acceptor with concomitant formation of H(2)O(2). In analogy to the thiol-disulfide oxidoreductase superfamily, a model for dithiol-disulfide exchange involving the conserved CxxC motif is proposed. Notably, while all studied disulfide oxidases invariably form intra- or interchenar disulfide bonds in peptides, GliT is the first studied enzyme producing an epidithio bond. Furthermore, through sensitivity assays using wild type, Delta gliT mutant, and complemented strain, we found that GliT confers resistance to the producing organism. A phylogenetic study revealed that GliT falls into a clade of yet fully uncharacterized fungal gene products deduced from putative ETP biosynthesis gene loci. GliT thus not only represents the prototype of ETP-forming enzymes in eukaryotes but also delineates a novel mechanism for self-resistance.

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

“…It has been demonstrated that A. fumigatus is capable of self protection against gliotoxin, and that deletion of gliT significantly increased strain sensitivity to exogenous gliotoxin (32,33). Both ⌬gliK mutants were also significantly more sensitive to gliotoxin (P Ͻ 0.001), and a reduced growth rate was seen at all time points, with the greatest difference in growth seen at 72 h (Fig.…”

“…Although a statistically significant difference in growth rates was observed between A. fumigatus wild-type and respective ⌬gliK strains, addition of gliotoxin did not completely suppress growth of ⌬gliK 46645 and ⌬gliK 26933 mutants, even at high concentrations of gliotoxin (20 g/ml). Thus, observed gliotoxin sensitivity was less apparent than that previously encountered for A. fumigatus ⌬gliT (32,33). Hence, we hypothesized that although gliK plays a role in self protection against gliotoxin, it is more likely that any metabolite (biosynthetic intermediate or shunt metabolite) produced consequent to gliK deletion mediates sensitivity to exogenous gliotoxin.…”

“…Glutathione (GSH) has been implicated as the immediate thiol source for both sulfur atoms in gliotoxin, and a reactive acyl imine intermediate has been proposed to act as an acceptor for glutathionylation catalyzed by the glutathione S-transferase GliG (11,31). The gliotoxin oxidoreductase, GliT, a key self-protection gene against gliotoxin, is regulated independently of gliZ and catalyzes disulfide bridge closure (32,33). The involvement of GliC has also been investigated (31), whereby GliC-mediated oxygenation of a gliotoxin biosynthetic intermediate was essential for subsequent thiolation catalyzed by GliG.…”

Quantitative proteomics reveals the mechanism and consequence of gliotoxin-mediated dysregulation of the methionine cycle in Aspergillus niger

“…Similarly at 6 h GT exposure, 26 GHs showed increased abundance compared to one that was detectable at lower levels (Table 3). In addition, peptidase abundance showed high levels of dysregulation, whereby 16 showed increased and 4 decreased abundance (at 3 h) and 18 increased with 8 showing reduced abundance at 6 h post-GT exposure ( Table 4). The majority of these degradative enzymes were observed at both timepoints (57.6% GHs and 43.8% peptidases).…”

Identification of Immunogenic Antigens from Aspergillus fumigatus by Direct Multiparameter Characterization of Specific Conventional and Regulatory CD4+ T Cells

“…The recombinant GliT protein (AFUA_6G09740) was produced in E. coli and purified as described (26). The open reading frames of the genes scw4 (AFUA_6G12380), aspf3 (AFUA_6G02280), shm2 (AFUA_3G09320), cpcB (AFUA_4G13170), and aspf22 (AFUA_6G06770) were amplified from ATCC46645 cDNA and cloned into the expression vector pET43.1H6 for recombinant expression as His-tagged proteins.…”